1. Field of the Invention
The present invention is directed to the killing of neoplastic cells. More specifically, the present invention relates to the use of folylpolyglutamyl synthetase (FPGS) gene transfer to enhance antifolate drug sensitivity.
2. Related Art
Because of the critical role of folate coenzymes in the synthesis of DNA precursors, folate antagonists (antifolates) have found widespread use as chemotherapeutic agents. Methotrexate (MTX), a 4-aminofolate analogue, has been in clinical use for the treatment of various human malignancies, especially leukemias and breast cancer, for about 40 years. For a review, see Chu, E. and Allegra, C, xe2x80x9cAntifolates,xe2x80x9d in Cancer Chemotherapy and Biotherapy: Principles and Practice, Chabner et al, eds., Lippincott-Raven, Philadelphia (1996), pp. 109-148.
MTX is a potent inhibitor of dihydrofolate reductase (DHFR). Inhibition of this enzyme prevents the reduction of dihydrofolate that accumulates in cells actively synthesizing thymaidylate via the thymidylate synthetase reaction. The cells subsequently become depleted of reduced folate cofactors, needed for synthesis of thymidylate and for de novo purine synthesis. The ensuing disruption of DNA replication leads to cell death in actively replicating cells found in tumors and some normal tissues.
Naturally occurring folates and some antifolates, including MTX, possess a single terminal benzoylglutamate residue and are converted intracellularly from monoglutamates into polyglutamates through the action of an enzyme, folylpolyglutamyl synthetase (FPGS), that attaches up to six glutamyl groups in xcex3-peptide linkage to the terminal benzoylgutamate. Polyglutamylation of folates and antifolates causes two effects. First, polyglutamylation causes intracellular accumulation of folates and antifolates because the highly ionized polyglutamylated forms are not readily transported across cell membranes. For example, MTX polyglutamates efflux out of cells 70 times slower than the monoglutamylated drug (Balinska, M., et al., Cancer Research 41:2751-2756 (1981)). Second, polyglutamylation enhances the affinity of folates for the enzymes that utilize them as cofactors, and increases the affinity (and inhibitory effect) of antifolates for their target enzymes, as well as expanding the range of enzymes which antifolates inhibit.
Because MTX is polyglutamylated much more inefficiently than naturally occurring folates, reductions in FPGS activity that have little effect on folate polyglutamate pools can have marked effects on the level of MTX polyglutamates and thus, on the cytotoxicity of MTX. The ability to generate MTX polyglutamates correlates directly with sensitivity to MTX for both human and murine tumor cells (Samuels, L. L., et al., Cancer Research 45:1488 (1985)). Human leukemia cell lines that have become resistant to clinically relevant antifolate doses through mutations in FPGS have been described (Pizzorno, G., et al., Cancer Research 48:2149 (1988); Roy, K., et al., Journal of Biological Chemistry 270:26918-26922 (1995); Roy, K., et al., Journal of Biological Chemistry 272:6903-6908 (1997); Takemura, Y., et al., British Journal of Cancer 75 (suppl. 1):31 (1997)). Human soft tissue sarcomas have been found to be intrinsically resistant to MTX as a result of low FPGS activity (Li, W. W., et al., Cancer Research 52:1434-1438 (1992)). Leukemias that have developed MTX resistance have been removed from patients and found to have impaired drug polyglutamylation (Rodenhuis, S., et al., Cancer Research 46:6513-6519(1986)). In addition, antifolates exhibiting the most therapeutic selectivity in murine tumor models consistently display a greater differential in accumulation of the polyglutamylated drug in tumor compared to normal proliferative tissues (Rumberger, S. et al., Cancer Research 50:4639-4643 (1990)).
Transfection of mutant CHO cells lacking FPGS activity with an FPGS expression cassette has been shown to increase the sensitivity of these cells to 4 hour MTX pulses in cell culture (Kim, J. S., et al., Journal of Biological Chemistry 268:21680-21685 (1993)). However, the question of whether tumor cells which already possess intermediate FPGS activity will show a similar enhancement of MTX sensitivity after FPGS gene delivery has not been previously addressed.
Traditional methods for cancer treatment rely on a combination of surgery, radiation, and cytotoxic chemotherapeutic drugs. Although the treatment of tumor cells with chemotherapeutic drugs is well-known in the art, presently, the therapeutic activity of many cytotoxic anti-cancer drugs is limited by a moderate therapeutic index associated with nonspecific toxicity toward normal host tissues, such as bone marrow, and the emergence of drug-resistant tumor cell sub-populations. One recent approach to enhancing the selectivity of cancer chemotherapeutics, and thereby reducing the toxicity of treatment, involves the application of gene therapy technologies to cancer treatment. See, Roth, J. A. and Cristiano, R. J., J. Natl. Cancer Inst. 89:21-39 (1997); Rosenfeld, M. E. and Curiel, D. T., Curr. Opin. Oncol. 8:72-77 (1996).
In one such therapy known in the art, the phenotype of the target tumor cells is genetically altered to increase the tumor""s drug sensitivity and responsiveness. One strategy being actively investigated involves directly transferring a xe2x80x9cchemosensitizationxe2x80x9d or xe2x80x9csuicidexe2x80x9d gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, F. L., Cancer Gene Therapy 1:279-287 (1994); Freeman, S. M., et al., Semin. Oncol. 23:3145 (1996); Deonarain, M. P., et al., Gene Therapy 2: 235-244 (1995)).
Several prodrug activation genes have been studied for application in cancer gene therapy. The two most extensively investigated prodrug-activating enzymes are herpes simplex virus thymidine kinase (HSV-TK), which activates the prodrug ganciclovir, and E. coli cytosine deazninase (CD), which activates the prodrug 5-fluorocytosine (Roth, J. A., Cristiano, R. J., Journal of the National Cancer Institute 89:21-39 (1997); Aghi, M., et al., Journal of the National Cancer Institute 90:370-380 (1998)).
HSV-TK phosphorylates the prodrug ganciclovir and generates nucleoside analogs that induce DNA chain termination and cell death in actively dividing cells. Tumor cells transduced with HSV-TK acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten, F. L. and Wells, J. M., J. Natl. Cancer Inst. 82:297-300 (1990); Ezzeddine, Z. D., et al., New Biol. 3:608-614 (1991).
The bacterial gene cytosine deaminase (CD) is a prodrug/enzyme activation system that has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent (Mullen, C. A., et al., Proc. Natl. Acad. Sci. USA 89: 33-37 (1992); Huber, B. E., et al., Cancer Res. 53:4619-4626 (1993); Mullen, C. A., et al., Cancer Res. 54:1503-1506 (1994)). Recent studies using these drug susceptibility genes have yielded promising results. See, e.g., Caruso, M., et al., Proc. Natl. Acad. Sci. USA 90:7024-7028 (1993); Oldfield, E., et al., Hum. Gene Ther. 4: 39 (1993); Culver, K, Clin. Chem 40: 510 (1994); O""Malley, Jr., B. W., et al., Cancer Res. 56:1737-1741 (1996); Rainov, N. G., et al., Cancer Gene Therapy 3:99-106 (1996).
Several other prodrug-activating enzyme systems have also been investigated (T. A. Connors, Gene Ther. 2:702-709 (1995)). These include the bacterial enzyme carboxypeptidase G2, which does not have a mammalian homolog, and can be used to activate certain synthetic mustard prodrugs by cleavage of a glutamic acid moiety to release an active, cytotoxic mustard metabolite (Marais, R., et al., Cancer Res. 56: 47354742 (1996)), and E. coli nitro reductase, which activates the prodrug CB 1954 and related mustard prodrug analogs (Drabek, D., et al., Gene Ther. 4:93-100 (1997); Green, N. K., et al., Cancer Gene Ther. 4:229-238 (1997)), some of which may be superior to CB1954 (Friedlos, F. et al., J Med Chem 40:1270-1275 (1997)). The principle underlying these approaches to prodrug activation gene therapy is that transduction of a tumor cell population with the foreign gene confers upon it a unique prodrug activation capacity, and hence a chemosensitivity which is absent from host cells that do not express the gene.
Current gene therapy technologies are limited by their inability to deliver prodrug activation or other therapeutic genes to a population of tumor cells with 100% efficiency. The effectiveness of this cancer gene therapy strategy can be greatly enhanced, however, by using drugs that exhibit a strong xe2x80x9cbystander effectxe2x80x9d (Pope, I. M., et al., Eur J Cancer 33:1005-1016 (1997)). Bystander cytotoxicity results when active drug metabolites diffluse or are otherwise transferred from their site of generation within a transduced tumor cell to a neighboring, naive tumor cell. Ideally, the bystander effect leads to significant tumor regression even when a minority of tumor cells is transduced with the prodrug activation gene (e.g., Chen, L., et al., Hum Gene Ther. 6:1467-1476(1995); Freeman, S., et al., Cancer Res. 53:5274-5283 (1993)). Bystander cytotoxic responses may also be mediated through the immune system, following its stimulation by interleukins and other cytokines secreted by tumor cells undergoing apoptosis (Gagandeep, S., et al., Cancer Gene Ther. 3:83-88 (1996)).
Although the ganciclovir/HSV-TK and 5-fluorocytosine/CD systems have shown promise in preclinical studies, and clinical trials are underway (Eck, S. L., et al., Hum Gene Ther. 7:1465-1482 (1996); Link, C. J. et al., Hum Gene Ther. 7:1161-1179 (1996); Roth, J. A., and Cristiano, R. J., J Natl Cancer Inst. 89:21-39 (1997)), several limitations restrict their efficacy and limit their application to cancer chemotherapeutics. These include: (a) the non-mammalian nature of the HSV/TK and CD genes, whose gene products may elicit immune responses that interfere with prodrug activation; (b) their reliance on drugs which were initially developed as antiviral drugs (ganciclovir) or antifingal drugs (5-fluorocytosine) and whose cancer chemotherapeutic activity is uncertain; (c) the dependence of these gene therapy strategies on ongoing tumor cell DNA replication; and (d) the requirement, in the case of HSV-TK, for direct cell-cell contact to elicit an effective bystander cytotoxic response (Mesnil, M., et al., Proc. Natl. Acad Sci. USA. 93: 1831-1835 (1996)). These considerations, together with the general requirement of combination chemotherapies to achieve effective, durable clinical responses, necessitates the development of alterative strategies to treat cancers using suicide gene-based (prodrug activation) gene therapy.
More recently, a drug activation/gene therapy strategy has been developed based on a cytochrome P450 gene (xe2x80x9cCYPxe2x80x9d or xe2x80x9cP450xe2x80x9d) in combination with a cancer chemotherapeutic agent that is activated through a P450-catalyzed monoxygenase reaction (Chen, L. and Waxman, D. J., Cancer Research 55:581-589 (1995); Wei, M. X., et al., Hum. Gene Ther. 5:969-978 (1994); U.S. Pat. No. 5,688,773, issued Nov. 18, 1997). Unlike the prodrug activation strategies mentioned above, the P450-based drug activation strategy utilizes a mammalian drug activation gene (rather than a bacterially or virally derived gene), and also utilizes established chemotherapeutic drugs (i.e., cyclophosphamnide) widely used in cancer therapy.
While MTX""s well-established chemotherapeutic activity also distinguishes it from prodrugs such as ganciclovir and 5-fluorocytosine, MTX possesses troublesome toxicity to normal tissues and its effectiveness could be improved by a gene transfer strategy that enhances the drug""s selective toxicity.
Thus, in light of the foregoing, there is a need in the art for a method that will enhance a neoplastic cell""s sensitivity to MTX or any other polyglutamylatable antifolate drug, and reduce the toxicity to normal tissues and cells.
The inventors have discovered that by introducing a FPGS gene (and thus an FPGS gene product) into neoplastic cells, the enzymatic conversion of an antifolate drug to its therapeutically active metabolites is greatly enhanced within the cellular and anatomic locale of the tumor, thereby increasing both the selectivity and efficiency with which neoplastic cells are killed. At the same time, undesirable side-effects to normal host cells are minimized.
The inventors first determined if transfection of an experimental brain tumor cell line with an expression cassette bearing the FPGS cDNA would increase the cells"" sensitivity to brief MTX pulses in culture. The ability of MTX to cause bystander killing of nontransfected tumor cells in cocultures of nontransfected and transfected cells was then ascertained. The next step involved determining if tumors formed by the transfected cells were more sensitive to reduced frequency of treatment in vivo than tumors formed by the nontransfected tumor cells. Finally, antifolates other than MTX were evaluated by treating the two cell lines and cocultures with brief pulses and by treating homogeneous and mixed tumors in vivo in order to determine what properties are desirable in drugs used in conjunction with FPGS gene delivery.
Accordingly, the present invention overcomes the disadvantages of the prior art by providing a method for killing neoplastic cells, the method comprising: (a) infecting the neoplastic cells with a vector for gene delivery, the vector comprising a folylpolyglutamyl synthetase (FPGS) gene; (b) treating the neoplastic cells with a chemotherapeutic agent that is activated by the product of the FPGS gene; and (c) killing the neoplastic cells.
The invention also provides a preferred embodiment of the foregoing methods wherein the FPGS gene is a mammalian gene, although the FPGS gene from any species could be used. The human FPGS gene is particularly preferred.
In another preferred embodiment of the foregoing methods, the FPGS-activated chemotherapeutic agent is a polyglutamylatable antifolate drug. Examples of such antifolate drugs include methotrexate (MTX), edatrexate (EDX), aminopterin, as well as antifolates which inhibit thymidylate synthetase. MTX and EDX are particularly preferred.
In another preferred embodiment of the foregoing methods, the neoplastic cells are malignant cells that are sensitive to antifolate chemotherapy, such as breast cancer and colon cancer. However, any neoplastic cell can be targeted since FPGS gene delivery will enhance the drug""s anticancer effect. Thus, neoplastic cells, such as, e.g., central nervous system tumors (gliomas, astocytomas), lymphomas, lung cancer, melanoma, pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, which are not typically treated with antifolate drugs, will be able to be used in the method of the invention.
The invention also provides a very particularly preferred embodiment of the foregoing methods, wherein the FPGS gene is the human FPGS gene and the chemotherapeutic agent is MTX.
The invention also provides a preferred embodiment of the foregoing methods, wherein the FPGS gene is delivered using a viral vector, preferably viral vectors whose use for gene therapy is well-established for those skilled in the art. Examples of such viral vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (including herpes simplex virus I and II and Epstein Barr virus), poliovirus, papillomavirus, or hybrid vectors having attributes of two or more viruses. Retroviruses, adenoviruses, and herpes viruses are particularly preferred viral vectors.
In another embodiment of the foregoing methods, the FPGS gene is delivered using any non-viral vector, preferably one whose use for gene therapy is well-established for those skilled in the art. Examples of such non-viral vectors for gene delivery include prokaryotic vectors (including tumor targeted bacterial vectors), cationic liposomes, DNA-protein complexes, non-viral T7 autogene vectors, fusogenic liposomes, direct injection of nucleic acid (xe2x80x9cnaked DNAxe2x80x9d), particle or receptor-mediated gene transfer, hybrid vectors such as DNA-adenovirus conjugates or other molecular conjugates involving a non-viral and viral component, starburst polyamidoamine dendrimers, cationic peptides, and mammalian artificial chromosomes.
In addition, the present invention provides an embodiment of the foregoing methods wherein the FPGS gene is delivered using any cellular vector, preferably one whose use for gene therapy is well-established for those skilled in the art. Examples of such cellular vectors for gene therapy include endothelial cells and macrophages including tumor-infiltrating macrophages, each of which may be modified using viral or non-viral vectors to carry the FPGS gene, and thus express the FPGS gene product.
The present invention also provides an embodiment, whereby the FPGS drug activation system is combined with another established gene/prodrug activation system, such as ganciclovir/HSV-TK and 5-fluorocytosine/CD. FPGS gene therapy may also be combined with other established cancer therapeutic genes, including tumor suppressor genes, such as p53; apoptotic factors, such as bax, tumor necrosis factor alpha, and caspases; and cytokines, such as interleukin 2, interleukin 4, and interleukin 12.
In another embodiment of the present invention, the targetting specificity for FPGS gene delivery is facilitated by xe2x80x9ctranscriptional targeting,xe2x80x9d including the use of tumor-specific or tumor-selective DNA enhancer sequences. Examples of such sequences include those described for genes that encode tyrosinase (melanoma), ERBB2 (pancreatic cancer), carcinoembryonic antigen (lung and gastrointestinal cancer), DF3/MUC1 (breast cancer), alpha-fetoprotein (hepatoma), as well as synthetic gene regulation systems which allow for transcriptional control and other forms of regulated expression of the FPGS gene. Targeting also includes sequences that control expression of genes induced by hypoxia (hypoxia response elements), or other tumor-specific conditions and factors.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.